Certain P-450 genes are under complex and poorly understood developmental control. Among the most interesting developmentally regulated P-450s is testosterone 7 alpha- hydroxylase (P-450a). To examine the mechanism by which P- 450a is controlled, we purified the enzyme from rat liver microsomes, produced specific polyclonal antibody and isolated its cDNA clone. The antibody and cDNA were used as probes to study levels of P-450a and its mRNA. Three mRNAs of approximately 2.0, 2.6 and 3.0 kilobase (Kb) in length hybridized with the P-450a cDNA probe in young adult male rats. Analysis of mRNA from 3-methylcholanthrene-treated rats revealed that only the 2.0 and 3.0 Kb mRNA were induced to a similar extent as the P-450a protein. In addition, levels of these two mRNAs correlated with levels of P-450a during development. The 2.6 Kb mRNA was only present in adult males and was absent throughout the life of females. These data suggest that the 2.0 and 3.0 Kb mRNAs are derived from the same gene via the use of different polyadenylation sites and that the 2.6 Kb mRNA is derived from a separate related differentially regulated gene. This supposition was confirmed by the isolation of two related genes from a rat gene library. The P-450a gene (2.0 and 3.0 Kb mRNAs) and the P- 450a2 gene (2.6 nucleotide mRNA) are highly homologous and both contain nine exons.